The long term goal of this research is to gain sufficient understanding of the immunogenicity and pathogenicity of Bordetella pertussis so that a safe, effective vaccine can be prepared. The goals of this proposal are to 1) isolate and characterize additional desirable antibodies for use as reagents. These antibodies will be monoclonal or highly specific for the adenylate cyclase, agglutinogens 1, 3, 4, and 6, and two outer membrane proteins, OMP36 and OMP22; 2) to use the new antibodies and existing antibodies to evaluate the antigen content of new acellular pertussis vaccine which is being prepared by the Department of Public Health, State of Michigan, under contract from the United States; and 3) to continue investigations into the nature of the antigens exposed on the surface of B. pertussis. In investigating surface antigens, we plan to study the phenomenon of the release of membrane vesicles, blebs, by B. pertussis during growth in broth. The blebs appear to be small packets of toxins and adhesins, and may be related to virulence. Since the blebs differ from the outermembrane in content of lipopolysaccharide and of several proteins, blebs may represent special domains on the outer membrane where virulence antigens are clustered together. Another possibility is that the adenylate cyclase is enzymatically active only when incorporated into a membrane, and that blebs may be a delivery system for the cyclase. Other surface antigens to be investigated include heat-modifiable proteins and peptidoglycan-associated proteins. We will also investigate the change in surface proteins which occurs in response to added nicotinic acid. This reversable shift, termed modulation, leads to loss of many surface proteins associated with virulence. We will also determine the change, if any, in surface antigens, of mutants altered in their virulence properties. This research is designed to take advantage of monoclonal antibodies, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and surface iodination of cells as techniques of probing the cell surface of B. pertussis.